Hormone-sensitive lipase (HSL) catalyzes the rate-limiting step in the mobilization of fatty acids from adipose tissue, thus determining the supply of energy substrates in the body. HSL mRNA was positively regulated by glucose in human adipocytes. Pools of stably transfected 3T3-F442A adipocytes were generated with human adipocyte HSL promoter fragments from −2,400/+38 to −31/+38 bp linked to the luciferase gene. A glucose-responsive region was mapped within the proximal promoter (−137 bp). Electromobility shift assays showed that upstream stimulatory factor (USF)-1 and USF2 and Sp1 and Sp3 bound to a consensus E-box and two GC-boxes in the −137-bp region. Cotransfection of the −137/+38 construct with USF1 and USF2 expression vectors produced enhanced luciferase activity. Moreover, HSL mRNA levels were decreased in USF1- and USF2-deficient mice. Site-directed mutagenesis of the HSL promoter showed that the GC-boxes, although contributing to basal promoter activity, were dispensable for glucose responsiveness. Mutation of the E-box led to decreased promoter activity and suppression of the glucose response. Analogs and metabolites were used to determine the signal metabolite of the glucose response. The signal is generated downstream of glucose-6-phosphate in the glycolytic pathway before the triose phosphate step.